1. Field of the Disclosure
The present invention relates generally to modified secreted viral proteins, to the genes which express these proteins and to antibodies produced against such proteins, and to the use of such materials in diagnostic and vaccine applications.
2. Brief Description of Related Art
Recombinantly produced proteins or fragments thereof are currently used in vaccines and immunodiagnostic assays. In order to purify and utilize recombinant protein products from eukaryotic cell expression systems, it is useful to engineer the system such that the molecules of interest are expressed at the highest possible level. Obviously, this is critical for commercial production. Secretion of the protein from cells into the medium is optimal and would eliminate many steps in the purification process, as well as eliminating losses of the material at each step.
Eukaryotic cells have a common mechanism for transporting secretory and some membrane proteins to their final destination utilizing a short predominantly hydrophobic signal peptide, usually near the N-terminal region of the protein in type I glycoproteins. This signal sequence is removed from the protein cotranslationally with the process of transit into the lumen of the endoplasmic reticulum. Membrane-bound proteins also contain a hydrophobic transmembrane anchor sequence within the molecule which, during migration of the protein from the inside to the outside of the cell, traps this region of the protein in the lipid bilayer of the membrane.
Viral glycoproteins can be isolated and purified from the lipid bilayer of cell membranes by solubilizing the membranes in detergent. However, this technique introduces problems with insoluble protein-detergent complexes, and protein-protein complexes which are not trivial to purify. Gething, et al., Nature 300:598-603 (1982) and later researchers have truncated viral proteins on the N-terminal side of the hydrophobic transmembrane anchor sequence (thus including the C-terminal cytoplasmic domain). Such a truncated protein secreted into the medium is already soluble and requires much less purification time and energy than an intracellularly expressed protein.
For the case of membrane glycoproteins, as opposed to cytoplasmic proteins, it is useful to engineer the secretion of a functional derivative into the medium rather than express the protein as a membrane-bound, cell-retained protein. Producing a secreted protein permits the development of continuous production processes in which a cell population is supplied with fresh medium daily in a continuous or episodic fashion and conditioned medium containing the protein of interest is withdrawn daily in a continuous or episodic fashion for recovery of the protein of interest.
Producing a secreted protein also facilitates recovery or purification of the protein for two reasons. First, the solubility of the protein is increased by the removal of the hydrophobic, lipophilic domains. Typically purification of membrane proteins requires the use and continuous presence of detergents, since the protein may only be soluble as a protein-detergent micellular complex, which makes the purification and formulation processes more difficult. Also, solubilization of some membrane glycoproteins requires the use of harsh detergents which denature glycoproteins and may result in their loss of functional activity. Second, cell culture can often be performed in the presence of medium with very low protein contents such that the initial relative concentration or specific activity of the protein of interest is much higher than if the protein is recovered from the cell fraction. Given the higher initial purity of the protein, the fold purification required to obtain a protein of high purity is much lower, fewer purification steps are required, and the resultant overall yield will be higher.